Limb salvage following massive segmental bone loss is a major challenge to the field of orthopaedics in both the military and civilian arenas. Critica bone defect surgeries require large devitalized segmental allograft transplantations to replace missing host bone segments, however significant problems often arise due to the impaired ability of the devitalized allograft to incorporate into the host bone. One of the most exciting strategies to promote and enhance allograft incorporation and critical bone defect healing involves the use of the patient's own bone marrow derived mesenchymal stromal/progenitor cells (MSCs). While this approach has demonstrated some preclinical success, problems remain in most cases including: inefficient MSC attachment to allograft via culturing techniques, uneven MSC distribution across the graft, as well as, weak adhesion of MSCs to the graft resulting in cell detachment in vivo. To overcome some of these problems, biodegradable polymer scaffolds have been widely used with cells to fabricate three-dimensional tissue-like grafts. However, strong inflammatory responses are often observed upon biodegradation of the scaffolds. Therefore, a new method to avoid the use of biodegradable scaffolds is strongly expected for allograft transplantation. Cell sheet technology utilizing temperature-responsive culture dishes has been applied to tissue engineering for several years to regenerate damaged tissues. Via this technology, cell sheets can be easily detached from culture dishes without any surface proteins and extracellular matrix (ECM) damage compared to cell sheets generated in standard cell culture dishes, and transplanted to the site of large bone defects, as a tissue-engineered periosteum surrounding the implanted graft. We expect that this new technique will significantly accelerate allograft incorporation into the host bone when compared to the use of allografts that have had MSCs directly seeded on the graft. Recently, we have successfully been able to isolate and culture human bone marrow derived MSCs. In this application, we will characterize the phenotypic changes caused by the cell sheet culturing techniques, and identify the optimal cell culture conditions necessary to generate MSC sheets using both freshly isolated human MSCs from patients and passaged MSCs (Lonza Inc.). Secondly, we will test the ability of human MSC sheets to repair a critical sized bone defect in the immuno-deficient mouse. The data generated by this proposal will likely identify a novel approach to generate a tissue-engineered periosteum to revitalize massive devitalized allografts used in critical segmented bone defect surgeries that will ultimately lead to improvements in the care and management of patients with large bone defects or other tissue damage.